X-linked Duchenne Muscular Dystrophy Research Articles

Sodium hydrosulfide moderately alleviates the hallmark symptoms of Duchenne muscular dystrophy in mdx mice

Duchenne muscular dystrophy (DMD) is an incurable disease caused by mutations in the X-linked DMD gene that encodes a structural muscle protein, dystrophin. This, in turn, leads to progressive degeneration of the skeletal muscles and the heart. Hydrogen sulfide (H2S), the pleiotropic agent with antioxidant, anti-inflammatory, and pro-angiogenic activities, could be considered a promising therapeutic factor for DMD.In this work, we studied the effect of daily intraperitoneal administration of the H2S donor, sodium hydrosulfide (NaHS, 100 μmol/kg/day for 5 weeks) on skeletal muscle (gastrocnemius, diaphragm and tibialis anterior) pathology in dystrophin-deficient mdx mice, characterized by decreased expression of H2S-generating enzymes.NaHS reduced the level of muscle damage markers in plasma (creatine kinase, lactate dehydrogenase and osteopontin). It lowered oxidative stress by affecting the GSH/GSSG ratio, up-regulating the level of cytoprotective heme oxygenase-1 (HO-1) and down-regulating the NF-κB pathway. In the gastrocnemius muscle, it also increased angiogenic vascular endothelial growth factor (Vegf) and its receptor (Kdr) expression, accompanied by the elevated number of α-SMA/CD31/lectin-positive blood vessels. The expression of fibrotic regulators, like Tgfβ, Col1a1 and Fn1 was decreased by NaHS in the tibialis anterior, while the level of autophagy markers (AMPKα signalling and Atg genes), was mostly affected in the gastrocnemius. Histological and molecular analysis showed no effect of H2S donor on regeneration and the muscle fiber type composition.Overall, the H2S donor modified the gene expression and protein level of molecules associated with the pathophysiology of DMD, contributing to the regulation of oxidative stress, inflammation, autophagy, and angiogenesis.

Clinical application of next generation sequencing-based haplotype linkage analysis in the preimplantation genetic testing for germline mosaicisms

BackgroundPreimplantation genetic testing (PGT) for monogenic disorders (PGT-M) for germline mosaicism was previously highly dependent on polymerase chain reaction (PCR)-based directed mutation detection combined with linkage analysis of short tandem repeats (STRs). However, the number of STRs is usually limited. In addition, designing suitable probes and optimizing the reaction conditions for multiplex PCR are time-consuming and laborious. Here, we evaluated the effectiveness of next generation sequencing (NGS)-based haplotype linkage analysis in PGT of germline mosaicism.MethodsPGT-M with NGS-based haplotype linkage analysis was performed for two families with maternal germline mosaicism for an X-linked Duchenne muscular dystrophy (DMD) mutation (del exon 45–50) or an autosomal TSC1 mutation (c.2074C > T). Trophectoderm biopsy and multiple displacement amplification (MDA) were performed for a total of nine blastocysts. NGS and Sanger sequencing were performed in genomic DNA of family members and embryonic MDA products to detect DMD deletion and TSC1 mutation, respectively. Single nucleotide polymorphism (SNP) sites closely linked to pathogenic mutations were detected with NGS and served in haplotype linkage analysis. NGS-based aneuploidy screening was performed for all embryos to reduce the risk of pregnancy loss.ResultsAll nine blastocytes showed conclusive PGT results. Each family underwent one or two frozen-thawed embryo transfer cycles to obtain a clinical pregnancy, and the prenatal diagnosis showed that the fetus was genotypically normal and euploid for both families.ConclusionsNGS-SNP could effectively realize PGT for germline mosaicism. Compared with PCR-based methods, the NGS-SNP method with increased polymorphic informative markers can achieve a greater diagnostic accuracy. Further studies are warranted to verify the effectiveness of NGS-based PGT of germline mosaicism cases in the absence of surviving offsprings.

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

Full-Length Dystrophin Restoration via Targeted Exon Addition in DMD-Patient Specific iPSCs and Cardiomyocytes.

Duchenne muscular dystrophy (DMD) is the most common fatal muscle disease, with an estimated incidence of 1/3500–1/5000 male births, and it is associated with mutations in the X-linked DMD gene encoding dystrophin, the largest known human gene. There is currently no cure for DMD. The large size of the DMD gene hampers exogenous gene addition and delivery. The genetic correction of DMD patient-derived induced pluripotent stem cells (DMD-iPSCs) and differentiation into suitable cells for transplantation is a promising autologous therapeutic strategy for DMD. In this study, using CRISPR/Cas9, the full-length dystrophin coding sequence was reconstructed in an exon-50-deleted DMD-iPSCs by the targeted addition of exon 50 at the junction of exon 49 and intron 49 via homologous-directed recombination (HDR), with a high targeting efficiency of 5/15, and the genetically corrected iPSCs were differentiated into cardiomyocytes (iCMs). Importantly, the full-length dystrophin expression and membrane localization were restored in genetically corrected iPSCs and iCMs. Thus, this is the first study demonstrating that full-length dystrophin can be restored in iPSCs and iCMs via targeted exon addition, indicating potential clinical prospects for DMD gene therapy.

Newborn screening for Duchenne muscular dystrophy-early detection and diagnostic algorithm for female carriers of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common pediatric-onset form of muscular dystrophy, occurring in 1 in 5,000 live male births. DMD is a multi-system disease resulting in muscle weakness with progressive deterioration of skeletal, heart, and smooth muscle, and learning disabilities. Pathogenic/likely pathogenic (P/LP) variants in the DMD gene, which encodes dystrophin protein, cause dystrophinopathy. All males with a P/LP variant in the X-linked DMD gene are expected to be affected. Two to 20% of female heterozygotes with a P/LP variant develop symptoms of dystrophinopathy ranging from mild muscle weakness to significant disability similar to Becker muscular dystrophy. Recently, with improvements in therapies and testing methodology, there is stronger evidence supporting newborn screening (NBS) for DMD for males and females because females may also develop symptoms. A consented pilot study to screen newborns for DMD was initiated in New York State (NYS) and conducted from 2019 to 2021. The identification of female carriers and the realization of the subsequent uncertainty of providers concerning follow-up during the pilot led to the development of algorithms for screening and diagnosis of carrier females, including both NBS and cascade molecular testing of family members.

EP463: Inconclusive Duchenne Muscular Dystrophy (DMD) carrier screening and atypical SNP-based NIPS sex chromosomes analysis suggest maternal sex chromosome abnormality

As of 2021, ACMG has expanded the recommended carrier screening panel to constitute 113 genes, now including the X-linked Duchenne Muscular Dystrophy (DMD) gene. Copy number variant (CNV) analysis by next-generation sequencing (NGS) of DMD yields inconclusive results for a small number of individuals due to the duplication/deletion of all DMD exons and adjacent regions on the X chromosome. In addition, a small number of SNP-based noninvasive prenatal screening (NIPS) tests yield no results for sex chromosomes due to a suspected maternal X chromosome abnormality.

DMD - TREATMENT: EP.146 scAAV.U7snRNA-mediated therapy: prolonged dystrophin expression and muscle function correction in adult Dup2 mice

Exon 2 duplications of the DMD gene, encoding the dystrophin protein, account for around 6-11% of all duplication mutations associated with X-linked Duchenne muscular dystrophy (DMD). As part of the preclinical development of a U7snRNA vector currently in a clinical trial (ClinicalTrials.gov NCT04240314), we have previously evaluated the therapeutic efficacy, absence of off-target splicing effects in AAV9.U7snRNA-mediated skipping of exon 2 in a murine Dmd model, and lack of toxicity in non-human primates. Here we report that 3-month-old Dup2 mice systemically injected with scAAV9.U7.ACCA vector, containing four copies of U7snRNA targeted to the exon 2 splice acceptor and splice donor sites, showed efficient exon 2 skipping, long-term dystrophin expression, and skeletal muscle function correction 18-months post vector administration. The RT-PCR data showed that a single vector injection (3E13 vg/kg) resulted in significant exon 2 skipping in tibialis anterior (TA), diaphragm (Dia) and heart tissues, showing an average of 46%, 32% and 73% total therapeutic transcripts, respectively. To determine the degree of functional rescue, in situ and in vitro physiology studies on TA and Dia muscles were performed. Both Dia and TA from 21-month-old control Dup2 mice exhibited a functional deficit with a significant reduction in specific force output (45-61%) compared with Bl6 mice. The significant force drop was also observed in those mice compared with Bl6 following a rigorous fatigue protocol. The single vector infusion resulted in a dramatic improvement in specific force output up to 64-76% in Dia and TA, and better protection of the TA muscle (up to 73%) from repeated fatigue. Overall, our results confirm that scAAV9.U7.ACCA provides long-term protection by restoring the disrupted dystrophin reading frame in straight muscles from Dup2 mice and functional recovery of TA and Dia muscles 18-month post vector administration.

Abstract P343: Physiologic Mechanical Stress In An Induced Pluripotent Stem Cell Derived Cardiomyocyte Model Of Duchene Muscular Dystrophy-related Cardiomyopathy Treated With A Membrane Resealant

Heart failure is leading cause of morbidity and mortality in the X-linked disease Duchenne muscular dystrophy (DMD). DMD is due to mutations the gene encoding dystrophin. Dystrophin localizes to the costamere in skeletal and cardiac muscle and is part of the larger dystrophin complex, which forms a critical connection linking the sarcomere to extracellular matrix. Disruptions in this complex lead to membrane fragility and multiple forms of muscular dystrophy, most of which have significant cardiac involvement. Therapeutic strategies for DMD include FDA-approved agents for exon skipping, as well as micro-dystrophins gene therapy, which is currently in clinical trials. Despite this progress, there is inadequate information as to how these and other novel agents will affect the DMD heart. Given the critical importance of cardiac muscle efficacy for any therapeutic for DMD and importance of membrane fragility in the disease phenotype, we assessed the susceptibility of patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring an out-of-frame exon 46-47 DMD deletion. iPSC-CMs were reprogrammed via a standard approach and differentiated, expanded, and purified by published methods. Troponin T flow cytometry was performed to and a minimum troponin T positivity >85% positivity was set for inclusion. DMD and control cells were plated onto flexible silicone membranes and subjected to equibiaxial strain as physiologic mechanical stressor, consistent with the inciting pathologic insult in DMD. Troponin and LDH release were assessed as clinically-relevant biomarkers of injury. Physiologic stress parameters were defined using troponin and LDH release relative to unstressed conditions. A membrane resealant being developed for treating DMD was shown to reduce troponin and LDH release in DMD iPSC-CMs, and also showed benefit in control cells. This work provides a ready platform for assessing therapeutics that target not only DMD-related cardiomyopathy, but other forms of cardiomyopathy and myocardial injury.

Early Myocardial Dysfunction and Benefits of Cardiac Treatment in Young X-Linked Duchenne Muscular Dystrophy Mice.

Duchenne muscular dystrophy (DMD) is associated with a progressive alteration in cardiac function. The aim of this study was to detect early cardiac dysfunction using the high sensitive two-dimensional speckle-tracking echocardiography (2D strain) in mdx mouse model and to investigate the potential preventive effects of the S107 ryanodine receptor (RyR2) stabilizer on early onset of DMD-related cardiomyopathy. Conventional echocardiography and global and segmental left ventricle (LV) 2D strains were assessed in male mdx mice and control C57/BL10 mice from 2 to 12months of age. Up to 12months of age, mdx mice showed preserved myocardial function as assessed by conventional echocardiography. However, global longitudinal, radial, and circumferential LV 2D strains significantly declined in mdx mice compared to controls from the 9months of age. Segmental 2D strain analysis found a predominant alteration in posterior, inferior, and lateral LV segments, with a more marked impairment with aging. Then, mdx mice were treated with S107 in the drinking water at a dose of 250mg/L using two different protocols: earlier therapy from 2 to 6months of age and later therapy from 6 to 9months of age. The treatment with S107 was efficient only when administered earlier in very young animals (from 2 to 6months of age) and prevented the segmental alterations seen in non-treated mdx mice. This is the first animal study to evaluate the therapeutic effect of a drug targeting early onset of DMD-related cardiomyopathy, using 2D strain echocardiography. Speckle-tracking analyses revealed early alterations of LV posterior segments that could be prevented by 4months of RyR2 stabilization.

X-linked Duchenne Muscular Dystrophy. Motor Functions and Prognosis

69 patients with x-linked Duchenne Muscular Dystrophy (DMD) were included in a retrospective investigation from 1975-1986. A mean profile of the decline of the motor functions was made by using the median age at which the patients were unable to perform specific motor functions by request. It was found that 81.2% of the personal profiles followed the pattern shown in the mean profile of the disease and that DMD is progressing in a uniform way, but with a different progression rate in every DMD patient. Good correlation between two motor functions made it possible to predict a personal prognosis of the decline of the motor functions in the DMD patient. No correlation between late walkers (later than 18 months) and rapid progression was found.

P.152A longitudinal study of miRNA biomarkers in paediatric Duchenne muscular dystrophy patients

Duchenne muscular dystrophy (DMD) is caused by mutations in the X-linked DMD gene leading to the absence or severe reduction of the dystrophin protein. In frame deletions in the DMD gene result in a production of truncated, partially functional protein and a milder phenotype of Becker muscular dystrophy (BMD). Although diagnosis and genetic confirmation for the type of DMD mutation, is not challenging per se, there is still a challenge for clinicians to predict the severity and the clinical course of the disease. With era of advanced gene therapy, requirement for specific follow-up biomarkers of the patients responses as well as predictive biomarkers is of outmost importance. MicroRNAs (miRNAs) are 19–22 nucleotides long non-coding RNAs that act as post-transcriptional regulators of gene expression and are used as biomarkers in different fields. In the context of DMD, there is a group of miRNAs called dystromirs, which are muscle specific and found to be elevated in serum of DMD patients. We here present the longitudinal study of previously validated biomarkers in 10 paediatric DMD and 5 BMD patients. We follow the expression levels of three miRNAs (miR-181a, miR-206 and miR-30c) during the three consecutive years and correlate their expression levels with standardised functional scores. We show that miR-206 expression is significantly higher in DMD patients compared to BMD patients during all three years. Thus, this miRNA may possibly distinguish between patients that will develop BMD phenotype to patients that will have DMD in early stages of the disease. In this long-term prospective follow-up, we demonstrate that both miR-206 and miR-30c are sensitive serum biomarkers for DMD regardless on the stage of the disease. We observe the trends of negative correlation of the biomarker levels with advancing age of the patients. We also report that the time required to run/walk 10 minutes and climb 4 stairs were the outcome measures that changed the most over time.

Use of Complementary and Alternative Medicine (CAM) in Patients with X-Linked Recessive Duchenne Muscular Dystrophy (DMD)

Use of Complementary and Alternative Medicine (CAM) in Patients with X-Linked Recessive Duchenne Muscular Dystrophy (DMD)

Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice

The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD.

Upregulation of brain utrophin does not rescue behavioral alterations in dystrophin-deficient mice

Dystrophin, the protein responsible for X-linked Duchenne muscular dystrophy (DMD), is normally expressed in both muscle and brain, which explains that its loss also leads to cognitive deficits. The utrophin protein, an autosomal homolog, is a natural candidate for dystrophin replacement in patients. Pharmacological upregulation of endogenous utrophin improves muscle physiology in dystrophin-deficient mdx mice, and represents a potential therapeutic tool that has the advantage of allowing delivery to various organs following peripheral injections. Whether this could alleviate cognitive deficits, however, has not been explored. Here, we first investigated basal expression of all utrophins and dystrophins in the brain of mdx mice and found no evidence for spontaneous compensation by utrophins. Then, we show that systemic chronic, spaced injections of arginine butyrate (AB) alleviate muscle alterations and upregulate utrophin expression in the adult brain of mdx mice. AB selectively upregulated brain utrophin Up395, while reducing expression of Up113 and Up71. This, however, was not associated with a significant improvement of behavioral functions typically affected in mdx mice, which include exploration, emotional reactivity, spatial and fear memories. We suggest that AB did not overcome behavioral and cognitive dysfunctions because the regional and cellular expression of utrophins did not coincide with dystrophin expression in untreated mice, nor did it in AB-treated mice. While treatments based on the modulation of utrophin may alleviate DMD phenotypes in certain organs and tissues that coexpress dystrophins and utrophins in the same cells, improvement of cognitive functions would likely require acting on specific dystrophin-dependent mechanisms.

238 TAILORED PIG MODEL OF DUCHENNE MUSCULAR DYSTROPHY

Duchenne muscular dystrophy (DMD) is one of the most common genetic diseases in humans, affecting 1 in 3500 boys. It is characterised by progressive muscle weakness and wasting due to mutations in the dystrophin (DMD) gene resulting in absence of dystrophin protein in skeletal muscle. Although curative treatments are currently not available, genetic and pharmacological approaches are under investigation including early-phase clinical trials. Existing animal models in different species (e.g. mdx mouse, GRMD dog) have been instrumental to understand the pathophysiology of DMD, but have several limitations. Importantly, the causative point mutations (mdx mouse: nonsense mutation; GRMD dog: splice mutation) are different from the most common human mutations (out-of-frame deletion of one or several exons of the DMD gene). We used gene targeting in somatic cells and nuclear transfer to generate a genetically tailored pig model of DMD. A bacterial artificial chromosome (BAC) from the porcine DMD gene was modified by recombineering to replace exon 52, resulting in a frame shift in the transcript. Modified BAC were transfected into male neonatal kidney cells, which were screened by quantitative polymerase chain reaction for replacement of exon 52 in the X-linked DMD gene. Eight of 436 cell clones were successfully targeted and 2 of them were used for nuclear transfer. For each of the cell clones, a pregnancy was established by transfer of cloned embryos into recipient gilts. Four piglets of the first litter were live born and killed within 48 h and tissue samples were processed for histological characterisation. Two piglets of the second litter died during birth due to obstetric complications, whereas the other 2 piglets were delivered by Caesarean section and raised in an artificial feeding system. Their serum creatine kinase (CK) levels were grossly elevated. Although both piglets showed reduced mobility compared with age-matched controls, they were able to move and feed on their own. Immunofluorescence staining of dystrophin was negative in muscle fibres of DMD mutant piglets and the complete absence of dystrophin protein was confirmed by immunoblot analysis. Histological examination of biceps femoris muscle from DMD mutant pigs showed a degenerative myopathy with fibre size variation, rounded fibres, central nuclei, fibrosis and fatty replacement of muscle tissue mimicking the hallmarks of the human disease. In conclusion, we generated the first pig model for a genetic muscle disease. The DMD mutant pig appears to be a bona fide model of the human dystrophy as ascertained by absence of the dystrophin protein, elevated serum CK levels and early degenerative changes on muscle histology. Because deletion of exon 52 is one of the most frequent mutations found in human DMD, the exon 52 mutated DMD pig represents an excellent model for testing targeted genetic treatments. This study was supported by the Bayerische Forschungsstiftung.

Altered presynaptic ultrastructure in excitatory hippocampal synapses of mice lacking dystrophins Dp427 or Dp71

Mental retardation is a feature of X-linked Duchenne muscular dystrophy (DMD) which likely results from the loss of the brain full-length (Dp427) and short C-terminal products of the dystrophin gene, such as Dp71. The loss of Dp427 or Dp71 is known to alter hippocampal glutamate-dependent synaptic transmission and plasticity in mice. Although dystrophins have a selective postsynaptic expression in brain, a putative role in retrograde regulation of transmitter release was suggested by studies in Drosophila. Here we used electron microscopy to analyze the distribution of synaptic vesicles in CA1 hippocampal axospinous non perforated-excitatory synapses of mice lacking Dp427 or Dp71 compared to control littermates. We found that the density of morphologically-docked vesicles is increased and the vesicle size is reduced in mice lacking Dp427, while in Dp71-null mice there is a decrease in the density of vesicles located in the vicinity of the active zone and an increase in the vesicle size and in the width of synaptic clefts. This is the first indication that the loss of mammalian brain dystrophins impacts on the presynaptic ultrastructural organization of central glutamatergic synapses, which may explain some of the alterations of synapse function and plasticity that contribute to intellectual disability in DMD.

Integrated DNA, cDNA, and protein studies in Becker muscular dystrophy show high exception to the reading frame rule

Becker muscular dystrophy (BMD) is a milder form of X-linked Duchenne muscular dystrophy (DMD). Here, we report a study of 75 patients with immunoblot and/or immunostaining findings of muscle biopsy consistent with BMD (abnormal dystrophin). We utilized multiplex ligation dependent probe amplification (MLPA) on genomic DNA (gDNA) to screen all 79 exons for both deletions and duplications. A total of 19 patients testing negative for MLPA mutations were tested for mRNA splicing abnormalities using cDNA-MLPA on muscle biopsy. Complete cDNA sequencing was done on MLPA-negative patients. We identified disease-causing mutations in 66 (88%) of the patients. Of the mutation-positive patients, 42 (64%) showed deletions of one or more exons, 14 (21%) showed duplications, and 10 (15%) showed various mutations detected by cDNA-MLPA and sequencing studies. We found a high rate of "exceptions" to the reading frame rule in this BMD series (out-of-frame BMD; 17/56 deletions/duplications; 30%). This was partly explained by the high incidence of 5' gene deletions in BMD patients (a region known to be a hotspot for exceptions), and due to complex splicing patterns in which a subset of transcripts showed deletions larger than gDNA (exon-skipping). Comparing our findings in BMD to previously published DMD data, BMD patients have higher proportions of duplications, a different distribution of mutations, and higher exception to the reading frame rule.

A and B utrophin in human muscle and sarcolemmal A-utrophin associated with tumours

Utrophin is an autosomal homologue of dystrophin, abnormal expression of which is responsible for X-linked Duchenne and Becker muscular dystrophy. In normal mature muscle utrophin is confined to blood vessels, nerves and myotendinous and neuromuscular junctions. When dystrophin is absent utrophin is abundant on the sarcolemma. This has raised the possibility that up-regulation of utrophin may be of therapeutic benefit. Two full-length transcripts of utrophin, A and B, have been identified, which are regulated by alternatively spliced 5′ promoters. In dystrophic mouse muscle, the A isoform is present on the sarcolemma, whereas the B form is confined to blood vessels. We show here using immunohistochemistry and human isoform-specific antibodies that A- and B-utrophin localisation is the same in human muscle. The A isoform is present on the sarcolemma of foetal human muscle fibres, regenerating fibres, fibres deficient in dystrophin and on blood vessels and neuromuscular junctions. B-utrophin is only detected on blood vessels. We also show that muscle adjacent to some soft tissue tumours shows increased sarcolemmal utrophin-A, showing that utrophin and dystrophin can simultaneously localise to the sarcolemma and raising the possibility that factor(s) from the tumour cells or accompanying inflammatory cells may have a role in regulating utrophin.

Muscular dystrophies and stem cells: a therapeutic challenge.

Correspondence to : E Gussoni, Division of Genetics, Children’s Hospital, 320 Longwood Ave, Boston, MA 02115, USA Introduction The muscular dystrophies are a diverse group of inherited disorders with common skeletal muscle pathological changes, characterized by progressive weakness and wasting due to muscle-cell necrosis [1,2]. Typical histological features include myofiber degeneration–regeneration, which lead to gradual destruction of skeletal muscle and replacement by connective tissue and fat. The X-linked Duchenne muscular dystrophy (DMD) represents the most frequent and severe form of muscular dystrophy, affecting 1 in 3500 live male births. Although the dystrophic process in DMD patients’ skeletal muscle is present at birth, the first motor difficulties do not appear until age 3–5 years. The disease leads to progressive loss of motor functions, wheelchair dependence by age 10–12 years and death in the third decade from respiratory and/or cardiac failures. In 1986–87, identification of the DMD gene and its encoded protein (dystrophin) opened new avenues of research [3–5]. Dystrophin is localized at the cytoplasmic side of the sarcolemma of skeletal and cardiac muscle [6], and is associated with a protein complex composed of trans-membrane (sarcoglycans, dystroglycans) and intracellular (dystrobrevins, syntrophins) proteins [7–10]. This dystrophin-associated protein complex (DAPC) provides a physical link between the actin cytoskeleton and the extracellular matrix, and one of its presumed functions is to stabilize the muscle-cell membrane during contraction– relaxation [7–10]. Since the discovery of the dystrophin gene, nearly 30 other forms of muscular dystrophy have been identified, many of them caused by mutations in members of the DAPC. For example, the limb-girdle muscular dystrophies (LGMD) LGMD2C-F result from mutations in one of four sarcoglycans [11,12]. Many of the histopathological changes observed in DMD are also seen in other forms of muscular dystrophy, implying that there are common pathways of muscle breakdown and regeneration among the different forms of dystrophy. Slowing or repairing these pathways may lead to effective disease treatment. In recent years, new mouse models have been generated for several forms of muscular dystrophy, including LGMDs [13]. These models not only have been very useful for the identification of common mechanisms underlying muscular dystrophy, but are also great tools for testing therapeutic approaches. In addition to these models, the naturally occurring muscular dystrophy models mdx (with a stop codon mutation in dystrophin exon 23) [14,15] and dy (with laminin a 2 chain deficiency) [16] are some of the most widely used models. Despite the progress made in understanding the molecular basis of muscular dystrophies, no efficient treatment is yet available for these devastating diseases [9,17,18]. Promising progress has been made in gene replacement [19,20] and pharmacological-based therapies, including utrophin up-regulation [21–23], corticosteroids [24–30] and aminoglycoside antibiotics [31,32]. However, this review will focus on recent efforts in the field of cell-based therapy.

Scoliosis in Duchenne muscular dystrophy

The x-linked Duchenne muscular dystrophy (DMD) is the most frequent generalized muscle disorder arising from a lack of the sarcolemmic protein "dystrophin". Patients with DMD develop in the majority a progressive scoliosis when they cease walking and/or standing at the age of 10 years and become confined to a wheelchair. Increasing muscle weakness leads to a progression of the curvature, the pelvic tilt and problems in sitting. Together with the simultaneous progressive weakness of the respiratory muscles a restrictive pulmonary insufficiency will occur. Surgical stabilization of the spine (> 20 degrees Cobb, forced vital capacity > 35%) by an adequate multisegmental instrumentation enabling early mobilization is now the treatment of choice. However, orthotic treatment may offer an acceptable compromise in exceptional cases, if the patient rejects surgical intervention or is in the late (inoperable) stages of the disease. Such a treatment is superior to a primary sitting support provision with insufficient possibilities of correction. The authors' experiences with 48 scoliosis orthoses made for 28 patients with DMD are reported. A "double plaster" cast has emerged as the best method to optimize adaption, especially in severe curvatures and the time taken for manufacturing the orthosis. A great deal of experience, patience and the consideration of the patients' individual demands are inevitable for a successful orthotic treatment.